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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Autoimmune Diseases

Autoimmune diseases are a group of disorders where the body's immune system malfunctions and attacks its own tissues. One aspect of these diseases is the formation of antibodies that are directed to self-antigens (autoantibodies). Autoimmune diseases can be divided into two general groups: Organ specific, where the autoantibodies attack a specific organ, and Non-organ specific (or systemic), where the autoantibodies attack multiple organ systems. An example of an organ specific autoimmune disease is Hashimoto thyroiditis where autoantibodies damage the thyroid gland. An example of a systemic autoimmune disease is systemic lupus erythematosus (SLE) where the autoantibodies may attack any organ in the body.

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Which of these is NOT considered one of the systemic autoimmune rheumatic diseases?View Page
Systemic Autoimmune Rheumatic Diseases

Systemic autoimmune rheumatic diseases (SARDs) are a group of autoimmune diseases that include:Rheumatoid arthritis (RA) Systemic lupus erythematosus (SLE) (and subsets of lupus) Sjögren syndrome (SjS) Systemic sclerosis (SSc) Polymyositis (PM) Dermatomyositis (DM)

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History of ANA Testing

Slide-based ANA testing using a cell substrate started in the 1950s and continues to be the gold standard method. In the early days of ANA testing, rodent tissue (stomach, liver and/or kidney) was commonly used as the substrate. Rodent tissue however had several drawbacks such as small cell size, a lack of dividing cells (mitotics) and poor antigen expression that made interpretation of ANA patterns difficult. In the 1980s, cultured cell lines were examined for utility as an ANA substrate and the human epithelial- like cell line HEp-2 gained popularity. HEp-2's advantages over rodent tissue are: A large nucleus Better antigen expression Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly).More recently a cell line called HEp-2000® has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This results in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.(Ref4)It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process.(Ref5) Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero.The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. Here they note the "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.(Ref6)

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Antigen Chart

Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA

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Homogeneous

This is an example of a homogeneous ANA pattern.This pattern is characterized by smooth staining in the nucleus of the interphase cells (a). The nucleoli may or may not stain. Notice the smooth staining in the chromosomal area of the metaphase mitotic cells (b).Follow-up testing for anti- dsDNA, histone or chromatin antibodies is recommended. These antibodies are primarily seen in patients with systemic lupus erythematosus (SLE).This pattern is reported as ANA positive, Homogeneous; titering is necessary.Homogeneous:Interphase cells Smooth staining of entire nucleus Nucleoli may or may not stainMetaphase mitotic cells Smooth staining of the chromosomal region No staining outside of the chromosomal area

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References

American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.

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Chemical Screening of Urine by Reagent Strip
Clinical Significance of Urine Protein

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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Examples of conditions resulting in benign proteinuria include: (Choose ALL correct answers)View Page

Chemical Screening of Urine by Reagent Strip (retired March 2012)
Clinical Significance cont'd

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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Proteinuria related to kidney impairment may be due to: (Choose ALL of the correct answers)View Page
Examples of conditions resulting in benign proteinuria include: (Choose ALL of the correct answers)View Page

Confirmatory and Secondary Urinalysis Screening Tests
Diseases Associated with Proteinuria

Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.

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Detecting and Evaluating Coagulation Inhibitors and Factor Deficiencies
Elevations in PT or aPTT assays

PT and/or aPTT may be prolonged for a number of reasons. Prolonged PT causes include: Warfarin therapy Liver disease Disseminated intravascular coagulation (DIC) Vitamin K deficiency Liver conditions such as cirrhosis or hepatitis Inadequate level of Factors I, II, V, VII, and/or XProlonged aPTT causes include: Presence of heparin Liver disease, other liver conditions Vitamin K deficiency Hemophilias DIC von Willebrand disease Lupus anticoagulant Inadequate levels of Factors I, II, V, VIII, IX, X, XI, and/or XII

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Interpreting the Mixing Study Results

Interpretation patterns for mixing study results include the following scenarios: If the results of the mixing study show correction for both the immediate and incubated PT/aPTT tests, the patient most likely has a factor deficiency (or multiple factor deficiencies). If the results of the mixing study show no correction in either the immediate or incubated PT/aPTT, the patient may have a coagulation inhibitor, most likely a lupus anticoagulant. If the results of the mixing study show correction for the immediate PT/aPTT results, but no correction in the incubated PT/aPTT, the patient may have a slow-acting inhibitor such as anti-factor VIII.Note: if the control tubes also show prolongation after incubation, there may have been a problem with the heat-labile factors in the sample losing their activity. If this is the case, the mixing study should be repeated.

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Lupus Anticoagulant

No single screening test can detect all lupus anticoagulant-positive (LA-positive) patients. Several tests are available and at least two should be employed to verify the presence of LA. Before any LA screening test is done, a thrombin time (TT) should be performed to rule out therapeutic heparin or the presence of a thrombin (factor-II) inhibitor.These are some of the LA screening procedures that can then be used: Dilute Russell's Viper Venom time (DRVVT). This test utilizes a reagent containing venom from the viper Vipera russelli (which activate factor V and X), low levels of phospholipids, and calcium ions in a clotting time test. The DRVVT test principle is based on the idea that the reagents can help to identify the antibody's dependence on phospholipids . Platelet neutralization procedure. This assay will show the dependence on phospholipids for the lupus anticoagulant to take effect. This can be performed using the aPTT based technique, with the DRVVT test, or using Taipan snake venom time tests. Kaolin clotting time or silica clotting time Hexagonal Phospholipid test (HPP). This is a similar assay to the platelet neutralization procedure, but thought to be more sensitive.

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Further Analyses for Coagulation Inhibitors (cont.)

When considering the presence of a coagulation factor inhibitor other than the common lupus anticoagulant, specific inhibitors such as anti-Factor VIII should be considered. Titers can be performed for the antibody in question to quantify the inhibitor and determine the extent of the antibody proliferation. These tests are called Bethesda titer assays. These tests use a universal inhibition unit in measuring specific-factor antibody activity. The appropriate dosing of medications may be measured based on these titer results.

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Further Analyses for Coagulation Inhibitors- Lupus Anticoagulant

For the diagnosis of lupus anticoagulant, the International Society on Thrombosis and Hemostasis has set a protocol of diagnostic criteria that should be met. This includes the following requirements: The patient sample must show abnormal phospholipid-dependent reactions in the coagulation lab. The patient sample must show inhibition of clotting after the mixing study test has been performed. The patient sample must be proven to have an inhibitor and not a factor deficiency. The patient must have a definitive phospholipid-dependent antibody and not a specific factor inhibitor.

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Treatment for Coagulation Inhibitors

Currently, there are several treatment options for patients suffering from coagulation inhibitors. Treat the patient by administering recombinant factor replacements. For example, using a recombinant factor VIII or factor IX for the treatment of acquired hemophilia due to coagulation inhibitors. Treat the patient with immunosuppresants, such as prednisone, to prevent large amounts of coagulation antibodies from forming. Then factor replacement can be given to the patient. If lupus anticoagulant is suspected, anticoagulants may be ordered to prevent thrombotic episodes. Often for treatment purposes, a patient is given a very high level of the coagulation factor that the antibody is targeting. The goal is to overwhelm the antibody with excess factor so that the antibody is neutralized and the residual factor can participate in the normal coagulation process.

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Introduction: Coagulation Inhibitors

As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.

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Coagulation Inhibitors and Coagulation Screening Tests

Patients with factor-specific coagulation inhibitors will have prolonged prothrombin time (PT) and/or aPTT test results (depending on the coagulation factor that is targeted by the inhibitor). Clinically, this is associated with abnormal clotting and bleeding complications. A prolonged aPTT, and sometimes PT, is seen with lupus anticoagulant. The antibody combines with the phospholipids on the surfaces of test reagents that are used in the aPTT test, and sometimes in the PT test, prolonging the test result(s). Clinically, lupus anticoagulant is associated with thrombosis and not with bleeding symptoms.Click here to read an important note regarding lupus anticoagulant

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References

1. Aniara Learning Center. Coagulation Corner. Mixing Studies: To correct or not correct-that is the question. June 2009. http://www.aniara.com/learning-center/Coagulation-Corner/articles/2009/01/mixing-studiesto-correct-or-not-correct.aspx.2. Bethel, M and Adcock, D: Laboratory evaluation of a prolonged APTT and PT. Lab Med, 285, May 2004.3. Devreese KM. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants. Thromb Res 2007;119:369-76.4. Harmening, D. Clinical Hematology and Fundamentals of Hemostasis. 5th edition. F.A. Davis, 2009.5. Katrien M.J. Devreese, Interpretation of normal plasma mixing studiesin the laboratory diagnosis of lupus anticoagulants, ThrombosisResearch, Volume 119, Issue 3, 2007, Pages 369-376, ISSN 0049-3848,DOI: 10.1016/j.thromres.2006.03.012.(http://www.sciencedirect.com/science/article/B6T1C-4JYKP68-1/2/12550b597f6b88b11e09b26e74963d4f)Keywords: Lupus anticoagulants; Mixing tests; Percent correction formula; Rosner index6. McKenzie, S. Clinical Laboratory Hematology. 2nd edition. Pearson, 2010.7. National Committee for Clinical Laboratory Standards. Determination of Factor Coagulant Activities, H48A. NCCLS, 1997.8. Santora SA, Eby CS, Chapter 106: Laboratory evaluation of hemostatic disorders. Pages 1841-1844. In: Hoffman R, Benz, EJ, Jr et. al Hematology. Basic Prinicples and Practice. 3rd edition. Churchill Livingstone. 2000.9. Vancott, E and Laposata, M: Coagulation, Fibrinolysis and Hypercoagulation. 2001.

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Emerging Cardiovascular Risk Markers
The hs-CRP Test

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

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General Laboratory Question Bank - Review Mode (no CE)
Cryoglobulin testing can be used to:View Page
Which of the following is not a feature of systemic lupus erythematosus (SLE):View Page

White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Erythrophagocytosis

Illustrated in the image is a phagocyte devouring several erythrocytes. This uncommon phenomenon occurs in the bone marrow and in the spleen as part of the process of erythrocyte destruction. Erythrophagocytosis is found in histological sections of the spleen in cases of hemolytic anemia. This phenomenon appears also in splenic sections in lupus erythematosis, and in rheumatoid arthritis. Our example is from a patient with a myeloproliferative disorder and is a rare example of a circulating erythrophagocytic cell in the peripheral blood.

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